Why is Fam83h critical for enamel formation?

为什么 Fam83h 对于牙釉质形成至关重要？

• 批准号: 7780358
• 负责人: JAN Ching Chun HU
• 金额: $ 36.33万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7780358
• 关键词: 8q24.3; Ameloblasts; Amelogenesis; Amelogenesis Imperfecta; Bacteria; Biochemical; Biological; Candidate Disease Gene; Cells; Characteristics; Chromosomes; Chromosomes, Human, Pair 8; Co-Immunoprecipitations; Code; Complex; Defect; Dental Enamel; Development; Disease; Dominant-Negative Mutation; Electron Microscopy; Enamel Formation; Etiology; Exons; Extracellular Matrix Proteins; Family; Genes; Genetic; Goals; Histologic; Immunohistochemistry; In Situ Hybridization; In Vitro; Inferior; Inherited; Lead; Learning; Link; MMP-20; Mass Spectrum Analysis; Monoclonal Antibodies; Mutate; Mutation; Nonsense Mutation; Odontogenesis; Oral health; Pain; Patients; Pattern; Peptides; Phenotype; Phosphoserine; Phosphothreonine; Phosphotyrosine; Play; Post-Translational Protein Processing; Principal Investigator; Proteins; Quality of life; Recombinants; Research; Role; Scanning; Secondary to; Structural Protein; Structure; System; Testing; Time Study; Tissues; Tooth structure; Transgenes; Transgenic Mice; Translations; Western Blotting; amelogenin; base; enamelin; genome-wide; glycosylation; in vivo; light microscopy; malformation; member; mouse model; novel; polyclonal antibody; programs; public health relevance; self esteem; yeast two hybrid system

DESCRIPTION (provided by applicant): Inherited diseases of dental enamel formation are grouped under the designation of amelogenesis imperfecta (AI). About 25% of all amelogenesis imperfecta (AI) cases are caused by the genes encoding four enamel extracellular matrix proteins, AMELX, ENAM, MMP20, and KLK4. Recently we identified mutations in FAM83H that cause AI in six different families suffering from amelogenesis imperfecta. This demonstrates that FAM83H is necessary for proper enamel formation. Virtually nothing is known about the Fam83h protein. Our objective is to learn the roles played by Fam83h in normal and defective enamel formation. Hypotheses: Because the phenotype in people with FAM83H mutations is limited to developing teeth, we hypothesize that Fam83H is expressed during odontogenesis. Because all of the AI- causing FAM83H mutations are dominant nonsense mutations that terminate translation in the last coding exon (exon 5), we further hypothesize that the Fam83h interacts with other cellular proteins to form functional complexes. We propose four Specific Aims: SA 1: To characterize the temporal and spatial pattern of Fam83h expression during odontogenesis and to determine its subcellular localization. SA 2: To isolate Fam83H protein for structural and functional characterization. SA 3: To identify Fam83H interacting proteins. SA 4: To determine if the expression of truncated Fam83h interferes with amelogenesis. Approach: The temporal and spatial pattern of Fam83h expression during odontogenesis is determined by in situ hybridization and immunohistochemistry. Recombinant and native Fam83h are used in structural and functional studies. Fam83h interacting proteins are identified using the yeast two-hybrid system and validated using a mammalian two-hybrid system, co- immunoprecipitation, and Far Western analyses. Normal and mutated Fam83h are expressed in ameloblasts as transgenes and their effects on enamel formation characterized. Heath Relatedness: Patients with inherited enamel defects (AI) have painful, disfigured teeth, lower self-esteem, and perceive themselves as having an inferior quality of life. The discovery that FAM83H is critical for dental enamel formation is a significant advance in our understanding of the etiology of AI. The proposed research may lead to a breakthrough in our understanding of important activities within ameloblasts and the discovery of new candidate gene(s) for AI. PUBLIC HEALTH RELEVANCE: Our group has determined that defects in FAM83H (family with sequence similarity 83 member H) on chromosome 8 cause severe enamel malformations (autosomal dominant hypocalcification amelogenesis imperfecta). Our objectives are to discover the normal function of Fam83h and how defects in this protein result in enamel malformations. Our long term goal is to advance understanding of normal and pathological tooth formation to stimulate improvements in the public's oral health.

描述（由申请人提供）：牙釉质形成的遗传性疾病被归类为釉质发育不全症（AI）。约25%的无釉发育不全（AI）病例是由编码4种釉质细胞外基质蛋白AMELX、ENAM、MMP20和KLK4的基因引起的。最近，我们在6个患有发育不全症的不同家族中发现了导致AI的FAM83H突变。这表明FAM83H对于牙釉质的形成是必需的。实际上，人们对Fam83h蛋白一无所知。我们的目的是了解Fam83h在正常和缺陷牙釉质形成中的作用。假设：由于FAM83H突变人群的表型仅限于发育中的牙齿，我们假设FAM83H在牙形成过程中表达。由于所有引起AI的FAM83H突变都是在最后一个编码外显子（外显子5）终止翻译的显性无义突变，我们进一步假设FAM83H与其他细胞蛋白相互作用形成功能复合物。我们提出了四个具体目标：1 .表征Fam83h在牙形成过程中的时空表达模式，并确定其亚细胞定位。SA 2：分离Fam83H蛋白进行结构和功能鉴定。SA 3：鉴定Fam83H相互作用蛋白。SA 4：确定截断的Fam83h的表达是否干扰成淀粉性发育。方法：采用原位杂交和免疫组织化学方法测定Fam83h在牙形成过程中的时空表达规律。重组和天然Fam83h用于结构和功能研究。Fam83h相互作用蛋白使用酵母双杂交系统鉴定，并使用哺乳动物双杂交系统、共免疫沉淀和Far Western分析进行验证。研究了正常和突变Fam83h基因在成釉细胞中的表达及其对釉质形成的影响。健康相关性：患有遗传性牙釉质缺陷（AI）的患者牙齿疼痛、变形，自尊心较低，并认为自己的生活质量较差。FAM83H对牙釉质形成至关重要的发现是我们对AI病因理解的重大进展。这项研究可能会导致我们对成釉细胞内重要活动的理解取得突破，并发现新的AI候选基因。